Showing posts from January, 2015

Considerations for immunofluorescence for various DREADD constructs

I receive frequent queries regarding the appropriate way to visualize DREADDs.  Below I show the three basic DREADD constructs:

HA-taggedmCherry fusion proteinsHA-tagged in a vector with IRES-mCitrene In unfixed tissue the mCherry and mCitrene can be visualized using the parameters from the table below or by going here.
NOTE: only the mCherry fusion protein fluorescence will show that the DREADD receptor is expressed.  mCitrene simply will show which cells were transfected or transduced.

For visualizing HA we have a detailed recommended protocol  the principle of which is below. I also show various secondary antibodies which might be used along with excitation/emission characteristics

Here's what you might consider if you have an mCherry fusion protein:

Finally, what if you are using one of the HA-hM3Dq-IRES-mCitrene or HA-hM4Di-IRES-mCitrene constructs.  Here you would want to use our HA staining protocol and then use an anti-mCitrene primary/secondary combination (one is shown bel…

Reversing (certain) autism deficits using DREADDs targeting Oxytocin neurons

Now here is a pretty amazing paper which has destroyed many of my objections related to the role of oxytocin and oxytocin neurons in mediating social behavior in mice (and potential for human therapeutics).

Paper just published in Science Translational Medicine....interesting ....

Also nice use of oxytocin promotor for cell-type specific expression of hM3Dq

New non-CNO actuators for DREADDs

Something we've been working on for a while now on-lineand were funded by the BRAIN Initiative.
These non-CNO compounds are available from Jian Jin.

Several of these will have significant advantages over CNO for moving the technology forward to primates including the approved medication perlapine (Hypnodin(R)) which shows >1000-fold selectivity for hM3Dq.

DREADDs as a tool to investigate addictive behavior

A timely review out summarizing what is known and what might be the potential of DREADD technology to elucidate and perhaps to treat addictive behaviors.

Restoration of learning with DREADDs

So frequently with opto- and chemogenetic technologies we see reports of extinction of various behaviors (or at least suppression).  Here is an interesting and well-controlled papershowing restoration of learning via cell-type-specific excitation with hM3Dq.

Heteromeric species

I frequently receive questions regarding the potential for DREADDs to form functional homo- and hetereomeric species.  There is a nice paper published some time ago indicating that at least in transfected cells any homomeric species are not of any significance in terms of signaling (e.g. a dimer between a DREADD and a native muscarinic receptor).

The broader question regarding potential hetereomeric species is unclear.  I note in passing that recent papers have reported failure to replicate D1/D2 hetereodimers and mGluR2/5-HT2A heterodimers suggesting that this field is still somewhat controversial.